A total of 879 subjects of Mulao ethnicity who reside in Luocheng Mulao Autonomous County (Guangxi Zhuang Autonomous Region, China) comprising 407 males (46.30%) and 472 females (53.70%) and 865 participants of Han Chinese ethnicity residing in the same location, including 425 males (49.13%) and 440 females (50.87%) were randomly selected from our previous stratified randomized samples. The age range was between 15 and 80 years. The mean age of the Mulao participants was 52.69±14.99 years, whereas that of the Han subjects was 52.29±14.26 years. All participants were essentially healthy rural agricultural workers, with no evidence of disease associated with atherosclerosis, CAD or diabetes. Any participant who had a history of taking medication known to affect serum lipid levels (lipid-lowering drugs, including statins or fibrates, β-blockers, diuretics or hormones) was excluded from the study prior to the blood sample being obtained. The study design was approved by the Ethics Committee of the First Affiliated Hospital, Guangxi Medical University (Nanning, China). Informed consent was obtained from all participants.

The survey was conducted using internationally standardized methods, following a previously described protocol (32). Information on demographics, socioeconomic status and lifestyle factors was collected with standardized questionnaires. Alcohol consumption was quantified as the number of liangs (~50 g) of rice wine, corn wine, rum, beer or liquor consumed during the preceding 12 months. Alcohol consumption was categorized into groups of grams of alcohol consumed per day: 0 (nondrinker), ≤25 and >25. Cigarette smoking status was categorized into groups of cigarettes per day: 0 (non-smoker), ≤20 and >20. At the physical examination, several parameters, including height, weight and waist circumference were measured. Sitting blood pressure was measured three times with the use of a mercury sphygmomanometer (Jiangsu Yuyue Medical Equipment & Supply Co., Ltd., Danyang, China) after at least 5 min of rest and the average of the three measurements was used to indicate blood pressure. Systolic blood pressure was determined by the first Korotkoff sound and diastolic blood pressure was determined by the fifth Korotkoff sound. Body weight, to the nearest 50 g, was measured with a portable balance scale. Height was measured, to the nearest 0.5 cm, using a stadiometer (Shanghai Sangon Biological Engineering Technology & Services Co., Shanghai, China). From these two measurements, body mass index (BMI, kg/m²) was calculated. Waist circumference was measured with a non-stretchable measuring tape.

Venous blood samples of 5 ml were drawn after at least 12 h of fasting. A total of 2 ml of the sample was collected into a glass tube and used to determine serum lipid levels. The remaining 3 ml was transferred to tubes with anticoagulants (4.80 g/l citric acid, 14.70 g/l glucose and 13.20 g/l trisodium citrate; Shanghai Sangon Biological Engineering Technology & Services Co.) and used to extract DNA. Measurements of serum TC, TG, HDL-C and LDL-C levels in the samples were performed using enzymatic methods with a Ransod autoanalyzer (Randox Laboratories Ltd., Crumlin, UK and Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan). Serum ApoA1 and ApoB levels were detected using the immunoturbidimetric immunoassay using a commercial kit (Randox Laboratories Ltd.). All determinations were performed with an autoanalyzer (Type 7170A; Hitachi Ltd., Tokyo, Japan) in the Clinical Science Experiment Center of the First Affiliated Hospital, Guangxi Medical University (27–30).

Genomic DNA of the samples was isolated from peripheral blood leukocytes using the phenol-chloroform method (27–30). The extracted DNA was stored at 4°C until analysis. Genotyping of the ANGPTL8 rs2278426 SNP was determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). PCR amplification was performed using the following primer sequences: Forward 5′-CAGGAGTTCTATTGTGCGGC-3′ and reverse 5′-CCTGATGCAACTATCGCACC-3′ (Shanghai Sangon Biological Engineering Technology & Services Co.). Each 25 µl PCR reaction mixture consisted of 2 µl genomic DNA, 1 µl each primer (10 pmol/l), 12.5 µl of 2X Taq PCR Master mix (constituent: 20 mM Tris-HCl, pH 8.3, 100 mM KCl, 3 mM MgCl₂, 0.1 U Taq Polymerase/µl, 500 µM dNTP each; Shanghai Sangon Biological Engineering Technology & Services Co.), and 8.5 µl of ddH₂O (DNase/RNase-free). PCR was performed with an initialization step of 95°C for 7 min, followed by 45 sec denaturing at 95°C, 45 sec of annealing at 60°C and 1 min of elongation at 72°C for 33 cycles. The amplification was completed with a final extension at 72°C for 7 min. Following electrophoresis on a 2.0% agarose gel with 0.5 µg/ml ethidium bromide, the amplification products were visualized under ultraviolet light. Subsequently, each restriction enzyme reaction was performed with 5 µl amplified DNA, 7.5 µl nuclease-free water, 1 µl of 10X buffer solution and 5 units BseGI (BtsCI) (Shanghai Sangon Biological Engineering Technology & Services Co.) restriction enzyme in a total volume of 15 µl, digested at 55°C overnight. Following restriction enzyme digestion of the amplified DNA, genotypes of the digestive products were identified using electrophoresis on 2% ethidium-bromide stained agarose gels and visualized under ultraviolet light. Genotypes were identified by an experienced technician blinded to the epidemiological and serum lipid results.

The six samples (AA, AG and GG genotypes in the two ethnic groups) detected by the PCR-RFLP were also confirmed by direct sequencing. The PCR product was purified by low melting point gel electrophoresis and phenol extraction and then the DNA sequences were analyzed by Shanghai Sangon Biological Engineering Technology & Services Co.

The normal values of serum TC, TG, HDL-C, LDL-C, ApoA1, ApoB levels and the ApoA1/ApoB ratio in our Clinical Science Experiment Center were 3.10–5.17, 0.56–1.70, 1.16–1.42, 2.70–3.10 mmol/l, 1.20–1.60, 0.80–1.05 g/l and 1.00–2.50, respectively. The individuals with TC>5.17 mmol/l and/or TG>1.70 mmol/l were defined as hyperlipidemic (33,34). Hypertension was diagnosed according to the criteria of the 1999 and 2003 World Health Organization-International Society of Hypertension Guidelines for the management of hypertension (35–37). The Cooperative Meta-analysis Group of China Obesity Task Force criteria were used for diagnosing an individual as overweight or obese (38). Normal weight, overweight and obese were defined as a BMI of <24, 24–28 or >28 kg/m², respectively (39).

Epidemiological data were recorded on a pre-designed form and managed with Microsoft Excel software (Microsoft, Redmond, WA, USA). All calculations were performed using SPSS software version 19.0 (IMB, Armonk, NY, USA) statistical package. The mean ± standard deviation (serum TG levels were presented as medians and interquartile ranges) and frequencies of baseline characteristics were calculated. Comparison of numerical variables, including age and body mass index, between the two groups was assessed using Student's unpaired t-test. Categorical variables were analyzed with a χ² test or Fisher's exact test. Allele frequency was determined via direct counting and the standard goodness-of-fit test was used to test the Hardy-Weinberg equilibrium. Differences in genotype distribution between the groups were calculated using the χ² test. The association of genotypes and serum lipid parameters were determined using analysis of covariance. Gender, age, BMI, blood pressure, alcohol consumption and cigarette smoking were adjusted for statistical analysis. Multivariate linear regression analysis with stepwise modeling was performed to evaluate the association of serum lipid levels with genotypes (GA/AA=1 and GG=2) and several environmental factors in the combined population and in, Mulao and Han groups, as well as males and females separately. P<0.05 was considered to indicate a statistically significant difference.

The comparison of general characteristics and serum lipid levels between the Mulao and Han populations is summarized in Table I. Body weight, BMI, cigarette smoking levels and diastolic blood pressure were all lower in the Mulao population than in the Han population (P<0.05-0.001), whereas the levels of ApoB were higher in the Mulao individuals than in the Han individuals (P<0.001) and the level of ApoA1 was higher in the Han individuals than in the Mulao individuals (P<0.05). No significant differences were identified in the levels of systolic blood pressure, serum TC, TG, HDL-C, LDL-C, the ApoA1/ApoB ratio, age structure, gender ratio, height and the amount of alcohol consumption between the two ethnic groups (P>0.05 for all).

After the genomic DNA of the samples was amplified using PCR and visualized with 2% agarose gel electrophoresis, products of 387 bp nucleotide sequences were observed in all samples (Fig. 1). The genotypes identified were termed according to the presence (A allele) or absence (G allele) of the enzyme restriction sites. Thus, the AA genotype is homozygous for the presence of the site (bands at 331 and 56 bp), the GA genotype is heterozygous for the presence and absence of the site (bands at 387, 331 and 56 bp) and the GG genotype is homozygous for the absence of the site (bands at 387 bp; Fig. 2). The 56 bp segment is not visible in the gel owing to its fast migratory speed. The genotypes of the rs2278426 SNP are in Hardy-Weinberg equilibrium.

The results are presented as GG, GA and AA genotypes by PCR-RFLP, and the GG, GA and AA genotypes were also confirmed by reverse sequencing (Fig. 3), respectively. The reverse sequence used as the SNP site was upstream of the sequence, therefore the same base pairs in a row in front of the PCR products influence forward sequencing.

The genotypic and allelic frequencies of the rs2278426 SNP in the ANGPTL8 gene are shown in Table II. The frequencies of G and A alleles were 82.20 and 17.80% in Han, and 76.96 and 23.04% in Mulao populations (P<0.001), respectively. The frequencies of GG, GA and AA genotypes were 68.79, 26.82 and 4.39% in the Han population and 60.64, 32.65 and 6.71% in the Mulao population (P<0.005), respectively. No differences in the genotypic frequencies between males and females in the two ethnic groups (P>0.05 for each) were identified.

As shown in Tables III and IV, serum LDL-C, ApoA1 and ApoB levels and the ApoA1/ApoB ratio in the Han but not in the Mulao population were different among the genotypes (P<0.05-0.001). The subjects with the GA/AA genotypes had lower LDL-C and ApoB levels, and higher ApoA1 levels and a higher ApoA1/ApoB ratio than the subjects with the GG genotype. No differences were observed in serum lipid levels between the genotypes in the Mulao population (P>0.05). Subgroup analyses revealed that serum TC, LDL-C, ApoB levels and the ApoA1/ApoB ratio in Han females but not in males were different among the genotypes (P<0.05-0.001), the subjects with the GA/AA genotype had lower TC, LDL-C and ApoB levels, and a higher ApoA1/ApoB ratio than the subjects with the GG genotype (P<0.05-0.001). No differences in serum lipid levels were identified between the specific genotypes in Mulao females or males (P>0.05 for each).

The multiple linear regression analysis revealed that LDL-C, ApoA1, ApoB levels and the ApoA1/ApoB ratio in the Han population were correlated with the specific genotypes (P<0.05-0.001; Table V). As shown in Table VI, when serum lipid data were analyzed according to gender, TC, LDL-C and ApoB levels and the ApoA1/ApoB ratio in Han females were correlated with specific genotypes (P<0.05-0.001). Serum lipid parameters were also associated with age, gender, BMI, systolic and diastolic blood pressure, fasting blood glucose levels, cigarette smoking and alcohol consumption in the two ethnic groups (P<0.05-0.001, Tables V and VI).